- 學位論文
利用 Bacillus sp. 相關菌株生產聚羥基丁酸(PHB)與聚羥基戊酸（PHV）共聚物及其性質探討
Production and characterization of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by Bacillus sp. strains
摘要
聚羥基烷酸(Polyhydroxyalkanoates, PHA)是許多細菌或真菌，如Ralstonia eutrophas、Alcaligenes latuss、Bacillus cereus、Pseudomonas pscudoflava，在過量碳源或缺少某些營養素（如氮、磷、硫）時，用以儲存能量的產物。本研究主要利用自酒精廢水中篩選出的數株本土菌株Bacillus sp. YW系列 ，探討是否具有累積PHA的能力。研究顯示，篩選出的Bacillus sp. YW系列菌株，皆可生產PHA。研究進一步證實：上述菌株皆具有代謝多種醣類與澱粉進行生長，進而合成PHB之能力。添加低濃度多種不同酸類時作為碳源時，會促進菌體的生長；在添加tryptone 情況，具有累積PHA產物的能力。初步研究顯示Bacillus sp. YW1與YW2在LBS培養基下可得菌體乾重與PHB 菌體含量達10 g/L與30%以上。 培養條件調整之後，使用LBSS培養基，在溫度30°C、轉速 200 rpm與起始酸鹼值pH=7時，可使Bacillus sp. YW1與YW2可誘導合成PHV，且PHV佔有菌體含量12%以上。TEM觀察也顯示菌體是具有累積PHA之能力。將培養時間36小時的純化的PHA由NMR與GC-MS鑑定，確認菌體生產的為PHBV。再由NMR的分析，可知兩菌株的PHBV中PHV含量最高可占有總量的莫耳分率約近30%。藉由GPC的分析，菌株YW1與YW2生產的PHA平均重量分子量集中在Mw=300,000-400,000之間，YW1的PDI為4.71與YW2的PDI為2.62；再藉由TGA分析，可確認菌株生產的PHBV有良好的熱穩定性。DSC分析顯示，純化之PHBV應為具有半結晶性質的高分子。
關鍵字
聚羥基丁酸,聚羥基戊酸,澱粉並列摘要
Polyhydroxylalkanoates (PHA) are biodegradable polyesters that are accumulated in various microorganisms as intracellular carbon and energy storage material under nutrient-limited conditions. This study aimed to screen better production strains in order to obtain more efficient production of Polyhydroxylalkanoates from Bacillus sp. YW1, Bacillus sp. YW2 and Bacillus sp. YW3. This studies shown that these strains could be improved to grow and further synthesize PHB using various carbon sources. This work also suggests that the bacterial growth could be improved which the medium contain various organic acids. On the other hand, these strains could accumulate PHA when adding tryptone in media. A semi-synthesized medium (LBS medium) was developed for this study which could be able to support the growth of this Bacillus sp. YW1 and Bacillus sp.YW2 with 10 g/L PHB production and PHB content over 30%, respectively. Temperature, pH and agitation rate are common environmental factors that play a crucial role in cell growth and PHB production. The optimal culture condition 30℃, pH 7, 200 rpm was used as the standard experimental condition. From this work, we found that the strains Bacillus sp. YW1 and YW2 could be induced to accumulate PHV over 12 %. PHA in the cell were purified primarily via different methods. The chemical structure and molecular masses of the purified PHA product were confirmed by nuclear magnetic resonance (1H-NMR & 13C-NMR) spectrometer and Gas Chromatography-Mass spectrometry (GC-MS), respectively. The property of the PHAs produced by Bacillus sp. strains isolated in this study was in consistent with the standard purchased from Sigma Co., indicating that Bacillus sp. strains are PHBV-synthesizing strain. Further analysis indicated that the PHV mole% of YW1 and YW2 strains were around 30%. According to analysis of GPC, the average Molecule weight was Mw=300,000 to 400,000; the strains YW1’s PDI was 4.71 and YW2’s PDI was 2.62. Besides, the data of TGA illustrated that PHBV has stable heat property. Moreover, the PHBV may be half-crystal polymer using DSC.